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Abstract:

The field of the present invention relates to genetically engineered
fusion molecules, methods of making said fusion molecules, and uses
thereof in anti-tumor immunotherapies. More specifically, the present
invention relates to engineered fusion molecules consisting of a tumor
targeting moiety fused with one or more costimulatory
molecules/chemokines/cytokines.

Claims:

1. A genetically engineered fusion molecule comprising a tumor targeting
moiety attached to a chemokine, wherein said fusion molecule exhibits
increased ADCC/tumor killing and enhanced activation of T cells at the
tumor site as compared to said tumor target moiety.

2. A fusion molecule of claim 1 wherein said tumor target moiety is an
antibody.

3. A fusion molecule of claim 2 wherein said antibody is an anti-her2/neu
antibody.

4. A fusion molecule of claim 3, wherein said chemokine is fractalkine.

5. A fusion molecule of claim 3, wherein said chemokine is MCP-1.

6. A fusion molecule of claim 1, wherein said tumor target moiety is
attached to said chemokine by a linker.

7. A fusion molecule of claim 1, further comprising a targeting peptide
fused to the tumor target moiety.

8. A pharmaceutical composition comprising a fusion molecule of claim 1 in
a pharmaceutically acceptable carrier.

9. A method for modulating an immune response in a patient, comprising:
administering to said patient a therapeutically effective amount of a
pharmaceutical composition of claim 8.

10. A method for treating tumors or tumor metastases in a patient,
comprising: administering to said patient a therapeutically effective
amount of a pharmaceutical composition of claim 8.

11. A genetically engineered fusion molecule comprising a tumor targeting
moiety attached to one or more costimulatory molecules, wherein said
fusion molecule delivers said costimulatory molecule to the tumor site
and promotes activation of T cells at the tumor site.

12. A fusion molecule of claim 11, further comprising a targeting peptide
fused to the tumor target moiety.

Description:

[0002]The field of the present invention relates to genetically engineered
fusion molecules, methods of making said fusion molecules, and uses
thereof in anti-tumor and anti-inflammatory immunotherapies. More
specifically, the present invention relates to engineered fusion
molecules consisting of a tumor or inflammatory cell targeting moiety
fused with one or more costimulatory molecules/cytokines and/or
chemokines. Importantly, the engineered fusion molecules of the present
invention provide focused immunological action to the disease site,
recruitment and activation of effector cytotoxic and NK cells, increased
target cell killing mediated by improved ADCC with the possibility of
demonstrating efficacy in patients with Fc receptor polymorphism, and
enhanced activation of T cells. As such, the novel fusion molecules
provide new and more effective immunotherapeutic approaches to a variety
of cancer and inflammatory diseases.

BACKGROUND OF THE INVENTION

[0003]Today, cancer remains a major cause of death and various diagnostic
and therapeutic methods for cancer have been developed. Immunotherapy is
the name given to cancer treatments that use the immune system to attack
cancers. Systemic immunotherapy refers to immunotherapy that is used to
treat the whole body and is more commonly used than local immunotherapy
which is used to treat one "localized" part of the body, particularly
when a cancer has spread. Although cancer cells are less immunogenic than
pathogens, the immune system is clearly capable of recognizing and
eliminating tumor cells, and cancer immunotherapy attempts to harness the
exquisite power and specificity of the immune system for treatment of
malignancy. Unfortunately, tumors frequently interfere with the
development and function of immune responses, i.e., the suppressive
milieu present within established tumors inhibits effective immune
responses. Thus, the challenge for immunotherapy is to use advances in
cellular and molecular immunology to develop strategies which manipulate
the local tumor environment to promote a proinflammatory environment,
promote dendritic cell activation, and effectively and safely augment
anti-tumor responses.

[0004]Conventionally, immunotherapy for cancers had previously been
centered on nonspecific immunotherapy. In recent years, however, it has
been clarified that T cells play an important role in tumor rejection in
living bodies. As a result, extensive efforts are now focused on T cell
responses and regulators of T cell activation. Targeted destruction of
malignancies by enhancing T cell responses is an attractive modality for
therapy because it potentially allows for exquisite specificity and
potent activity in the elimination of target cells while avoiding
toxicities associated with many other standard approaches.

[0007]Tumor antigen-specific, depleting antibody therapies deplete tumor
cells by antibody-directed cellular cytotoxicity (ADCC) and complement
dependent cytotoxicity (CDC). The ADCC is an immune effector mechanism
that requires: 1) therapeutic binding to the antigen through antibody
CDRs; and 2) antibody Fc binding to Fc receptors (FcR) expressed on
natural killer (NK) cells. Though the exact mechanism of ADCC function in
not known, two mechanisms have been postulated by scientists: 1) a
passive mechanism where FcR on effector cells serve as crosslinking
molecules; and 2) an active mechanism whereby activation of effector
cells leads to production and release of cytotoxic molecules such as
perforin (for pore formation) and granzyme B (for proteolysis). Various
defects have been associated with suboptimal response of ADCC. For
example, a correlation between the lack of drug response and an FcR
mutation has been established in many studies (Cartron et al., Blood,
99:754, 2002); Bowles and Weiner, JIM 304:88, 2005). Additional defects
include: (a) lack of NK cells and effector cytotoxic T cells recruitment
to the target site; (b) expression of killer inhibitory receptors (KIR)
on NK cells; and (c) expression of inhibitory Fc receptor.

[0008]Research efforts directed to resolving some of the issues associated
with antigen-specific cytotoxic T cells mediated therapies and the tumor
antigen-specific, depleting antibody therapies and been extensive. For
example, Holzer et. al. (U.S. Pat. No. 5,824,782) describe cancer
therapeutic agents comprising an antibody or antibody fragment specific
for the human epidermal growth factor receptor linked to a member of the
IL-8 chemokine family. The claimed immunoconjugates are shown to induce
cytotoxic and chemotactic activity and thus purported to be suitable for
targeted tumor therapy in human patients. There is no demonstration of in
vivo therapy using the immunoconjugates.

[0009]Lazar et al. (U.S. Pat. No. 7,317,091) describe and claim Fc
variants that are optimized for their ability to bind Fc gamma receptors
as compared to their parent polypeptide. The described Fc variants are
generally contained within a variant protein that preferably comprises an
antibody or Fc fusion protein. The Fc variants were reported to have
significant ADCC improvements.

[0010]Epstein et al., (U.S. Patent Application Nos. 20040228836 and
20070141025) disclose cancer therapeutic agents comprising a cancer
targeting molecule linked to a liver-expressed cytokine (LEC). The
preferred targeting molecule is an antibody specific for a tumor
cell-surface antigen, a stromal component of a tumor, or an intracellular
antigen. Importantly, only LECs are contemplated for use, and specific
LEC SEQ IDs are provided for such use.

[0011]The present invention is directed to resolving the issues above by:
1) improving antigen-specific cytotoxic T cells mediated therapies by
providing new and improved genetically engineered fusion molecules which
provide for focused delivery of a missing co-stimulatory
molecule/cytokine/chemokine to the tumor site to promote enhanced
recruitment and activation of effector T cells and NK cells; and 2)
improving tumor antigen-specific, depleting antibody therapies by
providing new and improved genetically engineered fusion molecules having
superior activity as compared to currently marketed drugs.

SUMMARY OF THE INVENTION

[0012]The present inventor seeks to improve on current antigen-specific
cytotoxic T cells mediated therapies. As such, one aspect of the present
invention is to provide a genetically engineered fusion molecule
comprising a cell/tumor targeting moiety fused to one or more
costimulatory molecules. In one embodiment, the fusion molecule will
comprise a tumor targeting moiety and a costimulatory molecule attached
to the tumor targeting moiety via a linker as depicted in any of the
FIGS. 1, 3 and 5. In another embodiment, the fusion molecule will further
comprise a targeting peptide attached to the tumor targeting moiety via a
linker as depicted in any of the FIGS. 2, 4, 6 and 7. In other
alternative embodiments, the fusion molecule may comprise a costimulatory
molecule and a cytokine attached via linkers to the tumor targeting
moiety. In particularly preferred embodiments, the targeting moiety will
be selected form the group consisting of, but not limited to, a depleting
antibody, Fab, Fab2, scFv, tumor binding peptide, or minimalistic
tumor/inflammatory cell binding domain; and the costimulatory molecule
will be selected from the group consisting of, but not limited to, one or
more of B7.1, B7.2, B7RP1, B7h, PD1, PDL1/PDL2, OX40L, CD86, CD40/CD40L
or 41BB/41BBL. Importantly, said fusion molecules deliver the missing
costimulatory molecule to the tumor site and promote optimal activation
of T cells. And because of the nature of the targeting moiety, focused
delivery of the signal is expected primarily to the tumor site. With
focused delivery and dose optimization, the fusion molecules of the
present invention are not expected to cause systemic activation of immune
system leading to autoimmunity as seen with some non-antigen specific
molecules currently in the clinical trials.

[0013]The present inventor also seeks to improve on existing tumor
antigen-specific, depleting antibody therapies. As such, another aspect
of the present invention is to provide a genetically engineered fusion
molecule comprising a cell/tumor targeting moiety fused to a chemokine.
In one embodiment, the fusion molecule will comprise a tumor targeting
moiety and a chemokine attached to the tumor targeting moiety via a
linker as depicted in any of the FIGS. 1, 3 and 5. In another embodiment,
the fusion molecule will further comprise a targeting peptide attached to
the tumor targeting moiety via a linker as depicted in any of the FIGS.
2, 4, 6 and 7. In other alternative embodiments, the fusion molecule may
comprise a chemokine and a cytokine attached via linkers to the tumor
targeting moiety. Importantly, said fusion molecules exhibit increased
ADCC and enhanced activation of T cells and/or NK cells at the tumor site
as compared to said tumor target moiety; addition of the cytokine will
serve to further enhance T cell recruitment to increase ADCC and promote
optimal activation of effector T cells.

[0014]Another aspect of the present invention relates to providing an
efficient and convenient method for preparing a genetically engineered
fusion molecule of the present invention. The method comprises the steps
of: 1) preparing/obtaining a cell/tumor targeting moiety; 2)
preparing/obtaining a costimulatory molecule and/or a chemokine and/or
cytokine; 3) preparing/obtaining a linker; 4) attaching 1) to 2) using
said linker to prepare a fusion molecule; and 5) purifying said fusion
molecule. Alternatively, the method may comprise, after step 4), step 5)
preparing/obtaining a targeting peptide; step 6) preparing/obtaining a
second linker; step 7) attaching the targeting peptide of step 5) to the
fusion molecule of step 4) using said second linker to prepare a fusion
molecule; and 8) purifying said fusion molecule.

[0015]Another aspect of the present invention relates to a pharmaceutical
composition, and method of preparing said pharmaceutical composition,
wherein said composition comprises the genetically engineered fusion
molecule of the present invention as an active ingredient, in a
pharmaceutically acceptable carrier.

[0016]Another aspect of the present invention relates to methods of
therapeutically treating a disease state in a subject. Such methods
include administering an effective amount of a genetically engineered
fusion molecule of the present invention in pharmaceutically acceptable
carrier to the subject, wherein such administration elicits an immune
response in a subject.

[0017]Another aspect of the present invention relates to a method of
treating tumors or tumor metastases in a patient, comprising
administering to said patient a therapeutically effective amount of a
genetically engineered fusion molecule of the present invention in
pharmaceutically acceptable carrier, wherein such administration promotes
tumor regression and/or tumor death.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 depicts one proposed design for a genetically engineered
fusion molecule of the present invention. In FIG. 1, the ovals labeled as
VL, VH, CL, CH1, CH2 and CH3 represent an example wherein the tumor
targeting agent is in the form of a full length antibody as defined
herein. The oval labeled C represents a cytokine, chemokine, or
costimulatory molecule. A linker is represented by the squiggled line. As
depicted in FIG. 1, C is attached to the tumor targeting agent via a
linker at the two VH sites. In one alternative embodiment, C will be
attached to the tumor targeting agent via a linker at the two VL sites
rather than the two VH sites. In yet another alternative, C will be
attached to the tumor targeting agent via a linker at the two CH3 sites
rather than two VL or two VH sites. Also contemplated are fusion
molecules wherein more than one C is attached to the targeting agent.

[0019]FIG. 2 depicts another proposed design for a genetically engineered
fusion molecule of the present invention. As in FIG. 1, the ovals labeled
as VL, VH, CL, CH1, CH2 and CH3 represent an example wherein the tumor
targeting agent is in the form of a full length antibody as defined
herein. The oval labeled C represents a cytokine, chemokine, or
costimulatory molecule, and a linker is represented by the squiggled
line. Further depicted in FIG. 2 are a targeting peptide (half
circle/arc) and a second linker (straight line). As depicted in FIG. 2,
the C is attached to the tumor targeting agent via a linker at the two VH
sites and the targeting peptide is attached via a linker at the two VL
sites. In one alternative embodiment, C will be attached to the tumor
targeting agent via a linker at the two VL sites and the targeting
peptide attached via a linker at the two VH sites. In yet another
alternative, C will be attached to the tumor targeting agent via a linker
at the two VH sites and the targeting peptide attached via a linker at
the two CH3 sites. In these designs, it is contemplated that the same
linker may be used for the C attachment and the targeting peptide
attachment.

[0020]FIG. 3 depicts another proposed design for a genetically engineered
fusion molecule of the present invention. In FIG. 3, the ovals labeled as
VL, VH, CH, CH1, and CH2 represent an example wherein the tumor targeting
agent is in the form of a Fab2 as defined herein. The oval label C
represents a cytokine, chemokine, or costimulatory molecule. A linker is
represented by the squiggled line. As depicted in FIG. 3, C is attached
to the tumor targeting agent via a linker at the two VH sites. In one
alternative embodiment, C will be attached to the tumor targeting agent
via a linker at the two VL sites rather than the two VH sites. In yet
another alternative, C will be attached to the tumor targeting agent via
a linker at the two CH2 sites rather than two VL or two VH sites. Also
contemplated are fusion molecules wherein more than one C is attached to
the targeting agent.

[0021]FIG. 4 depicts another proposed design for a genetically engineered
fusion molecule of the present invention. As in FIG. 3, the ovals labeled
as VL, VH, CL, CH1, and CH2 represent an example wherein the tumor
targeting agent is in the form of a Fab2 as defined herein. The oval
label C represents a cytokine, chemokine, or costimulatory molecule, and
the linker is represented by the squiggled line. Further depicted in FIG.
4 are a targeting peptide (half circle/arc) and a second linker (straight
line). As depicted in FIG. 4, the C is attached to the tumor targeting
agent via a linker at the two VH sites and the targeting peptide is
attached via a linker at the two VL sites. In one alternative embodiment,
C will be attached to the tumor targeting agent via a linker at the two
VL sites and the targeting peptide attached via a linker at the two VH
sites. In yet another alternative, C will be attached to the tumor
targeting agent via a linker at the two VH sites and the targeting
peptide attached via a linker at the two CH2 sites. In these designs, it
is contemplated that the same linker may be used for the C attachment and
the targeting peptide attachment.

[0022]FIG. 5 depicts another proposed design for a genetically engineered
fusion molecule of the present invention. In FIG. 5, the ovals labeled as
VL, VH, CL, and CH1 represent an example wherein the tumor targeting
agent is in the form of a Fab as defined herein. The oval label C
represents a cytokine, chemokine, or costimulatory molecule. A linker is
represented by the squiggled line. As depicted in FIG. 5, C is attached
to the tumor targeting agent via a linker at the VH site. In one
alternative embodiment, C will be attached to the tumor targeting agent
via a linker at the VL site. In yet another alternative, C will be
attached to the tumor targeting agent via a linker at the CH1 site. Also
contemplated are fusion molecules wherein more than one C is attached to
the targeting agent.

[0023]FIG. 6 depicts another proposed design for a genetically engineered
fusion molecule of the present invention. As in FIG. 5, the ovals labeled
as VL, VH, CL, and CH1 represent an example wherein the tumor targeting
agent is in the form of a Fab as defined herein. The oval label C
represents a cytokine, chemokine, or costimulatory molecule. A linker is
represented by the squiggled line. Further depicted in FIG. 6 are a
targeting peptide (half circle/arc) and a second linker (straight line).
As depicted in FIG. 6, C is attached to the tumor targeting agent via a
linker at the VH site and the targeting peptide attached via a linker at
the VL site. In one alternative embodiment, C will be attached to the
tumor targeting agent via a linker at the VL site and the targeting
peptide attached via a linker at the VH site. In yet another alternative,
C will be attached to the tumor targeting agent via a linker at the VH
site and the targeting peptide attached via a linker at the CH1 site. In
these designs, it is contemplated that the same linker may be used for
the C attachment and the targeting peptide attachment.

[0024]FIG. 7 depicts another proposed design for a genetically engineered
fusion molecule of the present invention. In FIG. 7, the ovals labeled as
CH2 and CH3 represent an example wherein the tumor targeting agent is in
the form of a peptide as defined herein. The oval label C represents a
cytokine, chemokine, or costimulatory molecule. A linker is represented
by the squiggled line. Further depicted in FIG. 7 are a targeting peptide
(half circle/arc) and a second linker (straight line). As depicted in
FIG. 7, C is attached to the tumor targeting agent via a linker at the
CH2 site and the targeting peptide attached via a linker at the CH3 site.
In another alternative, C will be attached to the tumor targeting agent
via a linker at the CH3 site and the targeting peptide attached via a
linker at the CH2 site. Alternatively, the tumor targeting peptide may be
linked to human serum albumin (HAS). In these designs, it is contemplated
that the same linker may be used for the C attachment and the targeting
peptide attachment.

DETAILED DESCRIPTION OF THE INVENTION

[0025]As those in the art will appreciate, the foregoing detailed
description describes certain preferred embodiments of the invention in
detail, and is thus only representative and does not depict the actual
scope of the invention. Before describing the present invention in
detail, it is understood that the invention is not limited to the
particular aspects and embodiments described, as these may vary. It is
also to be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to limit
the scope of the invention defined by the appended claims.

[0026]As used herein, an "antibody" refers to a protein comprising one or
more polypeptides substantially or partially encoded by immunoglobulin
genes or fragments of immunoglobulin genes. The recognized immunoglobulin
genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu
constant region genes, as well as myriad immunoglobulin variable region
genes. Light chains are classified as either kappa or lambda. Heavy
chains are classified as gamma, mu, alpha, delta, or epsilon, which in
turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively. A typical immunoglobulin (e.g., antibody) structural unit
comprises a tetramer. Each tetramer is composed of two identical pairs of
polypeptide chains, each pair having one "light" (about 25 kD) and one
"heavy" chain (about 50-70 kD). The N-terminus of each chain defines a
variable region of about 100 to 110 or more amino acids primarily
responsible for antigen recognition. The terms variable light chain (VL)
and variable heavy chain (VH) refer to these light and heavy chains,
respectively.

[0027]In a full-length antibody, each heavy chain is comprised of a heavy
chain variable region (abbreviated herein as HCVR or VH) and a heavy
chain constant region. The heavy chain constant region is comprised of
three domains, CH1, CH2 and CH3. Each light chain is comprised of a light
chain variable region (abbreviated herein as LCVR or VL) and a light
chain constant region. The light chain constant region is comprised of
one domain, CL. The VH and VL regions can be further subdivided into
regions of hypervariability, termed complementarity determining regions
(CDR), interspersed with regions that are more conserved, termed
framework regions (FR). Each VH and VL is composed of three CDRs and four
FRs, arranged from amino-terminus to carboxy-terminus in the following
order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can
be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g.,
IgG1, IgG2, IgG 3, IgG4, IgA1 and IgA2) or subclass.

[0028]The term "Fc region" is used to define the C-terminal region of an
immunoglobulin heavy chain, which may be generated by papain digestion of
an intact antibody. The Fc region may be a native sequence Fc region or a
variant Fc region. The Fc region of an immunoglobulin generally comprises
two constant domains, a CH2 domain and a CH3 domain, and optionally
comprises a CH4 domain. Replacements of amino acid residues in the Fc
portion to alter antibody effector function are known in the art (see,
e.g., Winter, et al., U.S. Pat. Nos. 5,648,260; 5,624,821). The Fc
portion of an antibody mediates several important effector functions e.g.
cytokine induction, ADCC, phagocytosis, complement dependent cytotoxicity
(CDC) and half-life/clearance rate of antibody and antigen-antibody
complexes.

[0029]The term "monoclonal antibody" as used herein refers to an antibody
obtained from a population of substantially homogeneous antibodies, i.e.,
the individual antibodies comprising the population are identical except
for possible naturally occurring mutations that may be present in minor
amounts. Monoclonal antibodies are highly specific, being directed
against a single antigen. Furthermore, in contrast to polyclonal antibody
preparations that typically include different antibodies directed against
different determinants (epitopes), each monoclonal antibody is directed
against a single determinant on the antigen. The modifier "monoclonal" is
not to be construed as requiring production of the antibody by any
particular method.

[0030]The term "human antibody", as used herein, is intended to include
antibodies having variable and constant regions derived from human
germline immunoglobulin sequences. The human antibodies of the invention
may include amino acid residues not encoded by human germline
immunoglobulin sequences (e.g., mutations introduced by random or
site-specific mutagenesis in vitro or by somatic mutation in vivo), for
example in the CDRs and in particular CDR3. However, the term "human
antibody", as used herein, is not intended to include antibodies in which
CDR sequences derived from the germline of another mammalian species,
such as a mouse, have been grafted onto human framework sequences.

[0031]The term "recombinant human antibody", as used herein, is intended
to include all human antibodies that are prepared, expressed, created or
isolated by recombinant means, such as antibodies expressed using a
recombinant expression vector transfected into a host cell; antibodies
isolated from a recombinant, combinatorial human antibody library;
antibodies isolated from an animal (e.g., a mouse) that is transgenic for
human immunoglobulin genes; or antibodies prepared, expressed, created or
isolated by any other means that involves splicing of human
immunoglobulin gene sequences to other DNA sequences. Such recombinant
human antibodies have variable and constant regions derived from human
germline immunoglobulin sequences. In certain embodiments, however, such
recombinant human antibodies are subjected to in vitro mutagenesis (or,
when an animal transgenic for human Ig sequences is used, in vivo somatic
mutagenesis) and thus the amino acid sequences of the VH and VL regions
of the recombinant antibodies are sequences that, while derived from and
related to human germline VH and VL sequences, may not naturally exist
within the human antibody germline repertoire in vivo. All such
recombinant means are well known to those of ordinary skill in the art.

[0032]The present invention relates to genetically engineered fusion
molecules comprising at least one tumor targeting moiety linked to at
least one costimulatory molecule (or at least one chemokine or cytokine)
formed through genetic fusion or chemical coupling. By "linked" we mean
that the first and second sequences are associated such that the second
sequence is able to be transported by the first sequence to a target
cell, i.e., fusion molecules in which the tumor targeting moiety is
linked to a costimulatory molecule (or chemokine or cytokine) via their
polypeptide backbones through genetic expression of a DNA molecule
encoding these proteins, directly synthesized proteins, and coupled
proteins in which pre-formed sequences are associated by a cross-linking
agent. In one embodiment the tumor targeting moiety and costimulatory
molecules (or chemokine or cytokine) are linked directly to each other
using recombinant DNA techniques. In another embodiment, the tumor
targeting moiety and costimulatory molecules (or chemokine or cytokine)
are linked via a linker sequence. The term "attached" as used herein
refers to such linkages/fusions.

[0035]Preferred tumor targeting moieties contemplated for use in the
fusion molecules of the present invention include depleting antibodies to
specific tumor antigens, including, but not limited to, anti-Her2/neu,
anti-Her3, anti-Her4, anti-CD20, anti-CD19, anti-CD22, anti-CXCR3,
anti-CXCR5, anti-CCR3, anti-CCR4, anti-CCR9, anti-CRTH2, anti-PMCH,
anti-CD4, and anti-CD25. All such tumor and inflammatory cell-specific,
depleting antibodies have been well described in the literature.

[0036]The term "costimulatory molecule", as used herein, is intended to
refer to a group of immune cell surface receptor/ligands which engage
between T cells and antigen presenting cells and generate a stimulatory
signal in T cells which combines with the stimulatory signal (i.e.,
"co-stimulation") in T cells that results from T cell receptor ("TCR")
recognition of antigen on antigen presenting cells, i.e., its art
recognized meaning in immune T cell activation. Also contemplated for use
are soluble forms of costimulatory molecules, i.e., those costimulatory
molecules normally expressed by B cells, macrophages, monocytes,
dendritic cells and other such antigen presenting cells. Costimulatory
molecules contemplated for use thus include, but are not limited to, one
or more of B7.1, B7.2, B7RP1, B7h, PD1, PDL1/PDL2, OX40L, CD86,
CD40/CD40L or 41BB/41BBL. The choice of which costimulatory molecule to
include in a particular embodiment depends upon, e.g., which particular
immune response effects are desired, e.g., a humoral response, or a
cellular immune response, or both. In certain embodiments both cellular
and humoral immune responses against a disease related antigen are
desired, and fusion molecules with varying costimulatory molecule domains
are contemplated for use. Because of the nature of the targeting moiety,
focused delivery of the signal is expected primarily to the tumor site.
With focused delivery and dose optimization, the fusion molecules of the
present invention are not expected to cause systemic activation of immune
system leading to autoimmunity as seen with some non-antigen specific
molecules currently in the clinical trials.

[0037]Chemokines are a superfamily of small (approximately about 4 to
about 14 kDa), inducible and secreted pro-inflammatory cytokines that act
primarily as chemoattractants and activators of specific leukocyte cell
subtypes. Their production is induced by inflammatory cytokines, growth
factors and pathogenic stimuli. Chemokine signaling results in the
transcription of target genes involved in motility, cell invasion, and
interactions with the extracellular matrix (ECM). Migration of cells that
express the appropriate chemokine receptor occurs along the concentration
gradient of the ligand known as the chemokine gradient; moving from a
lower to higher concentration. Structural analysis demonstrates that most
chemokines function as monomers and that the two regions necessary for
receptor binding reside within the first 35 amino acids of the flexible
N-terminus (Clark-Lewis et al., J Leukoc Biol. 57:703-11, 1995; Beall et
al., Biochem J 313:633-40, 1996).

[0038]The chemokine proteins are divided into subfamilies based on
conserved amino acid sequence motifs and are classified into four highly
conserved groups--CXC, CC, C and CX3C, based on the position of the first
two cysteines that are adjacent to the amino terminus. To date, more than
50 chemokines have been discovered and there are at least 18 human
seven-transmembrane-domain (7TM) chemokine receptors. In general, these
receptors, which belong to the G-protein-coupled receptor (GPCR) family,
often bind to more than one type of chemokine. There are six
non-promiscuous receptor-ligand pairs known to date--CXCR4-SDF1,
CXCR5-CXCL13, CXCR6-CXCL16, CCR6-CCL20, CCR9-CCL25 (also known as TECK)
and CX3CR1-CX3CL1 (also known as fractalkine or FKN).

[0039]The alpha subfamily (the CXC chemokines) has one amino acid
separating the first two cysteine residues. The receptors for this group
are designated CXCR1 to CXCR6. In the beta subfamily (the CC chemokines)
the first two cysteines are adjacent to one another with no intervening
amino acid. There are currently 24 distinct human beta subfamily members.
The receptors for this group are designated CCR1 to CCR11. Target cells
for different CC family members include most types of leukocytes. In the
gamma subfamily (C chemokine), the chemokine protein contains two
cysteines, corresponding to the first and third cysteines in the other
groups. Lymphotactin is the lone member of the gamma class. The
lymphotactin receptor is designated XCR1. In the delta subfamily (CX3C
chemokine), the protein has three intervening amino acid between the
first two cysteine residues. Fractalkine (FKN) is the only known member
of the delta class. The fractalkine receptor is known as CX3CR1.

[0040]In a particularly preferred embodiment of the present invention, the
chemokine will be FKN. This molecule is unique among chemokines in that
it is a transmembrane protein with the N-terminal chemokine domain fused
to a long mucin-like stalk. This membrane-anchored localization of FKN
has led to the suggestion that it functions as a cell adhesion molecule
for circulating inflammatory cells. Data supporting this hypothesis have
come from numerous in vitro experiments showing that immobilized FKN,
either on glass substrata or monolayers of CX3CR1 transfected cells, can
support the capture and adhesion of leukocytes. These adhesive functions
of FKN appear to be mediated by a single GPCR, CX3CRI, expressed on
monocytes, DCs, NK cells, neurons, microglia and effector T-cells. In
addition to functioning as an adhesion molecule, FKN can be released from
the cell surface by a protease such as TACE(ADAM17) to generate a soluble
molecule that has chemotactic activity for cells bearing the CX3CR1
receptor. By engineering a molecule with specificity to antigen (e.g.
tumor antigen or a molecule overexpressed in pathological state)
genetically fused with a chemokine ligand (full length, mutated for
enhanced or dominant negative activity, truncated as to act for enhanced
or dominant negative activity, or modified for enhanced or dominant
negative activity) such as FKN will create chemokine gradient around
tumor and therefore allow chemokine receptor expressing cells will
migrate to tumor. Among many chemokine receptors, CX3CR1 is highly
expressed on NK cells and effector cytotoxic T cells. Both of these cell
types are rich in molecules needed for cell death, i.e. perforin and
granzyme. By bringing NK cells and effector cytotoxic T cells closer to
tumor through FKN we expect the following activities: (i) increased ADCC
(ii) costimulation and thus adequate activation of effector cytotoxic T
cells (iii) efficacy in patients with FcR mutation, i.e., retreatment
opportunities for patients who previously failed to respond to antibody
monotherapy. Other chemokines contemplated for use include, but are not
limited to, MIP1a and MIP1.

[0041]In certain embodiments of the present invention, either the N- or
C-terminus of antibody heavy or light chain will be genetically
constructed with one of the several contemplated costimulatory ligands
and/or chemokine. In other embodiments of the present invention, a
targeting peptide or cytokine may be added to unused N- or C-terminus of
antibody heavy or light chain to further enhance recruited cells
activation in a tissue targeted fashion. The term "cytokine" is a generic
term for proteins released by one cell population, which act on another
cell population as intercellular mediators. Examples of such cytokines
are lymphokines, monokines, and traditional polypeptide hormones.
Included among the cytokines are growth hormone such as human growth
hormone, N-methionyl human growth hormone, and bovine growth hormone;
parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin;
glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid
stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth
factor; fibroblast growth factor; prolactin; placental lactogen; tumor
necrosis factor-alpha and -beta; mullerian-inhibiting substance; mouse
gonadotropin-associated peptide; inhibin; activin; vascular endothelial
growth factor; integrin; thrombopoietin (TPO); nerve growth factors such
as NGF-alpha; platelet-growth factor; transforming growth factors (TGFs)
such as TGF-alpha and TGF-beta; insulin-like growth factor-1 and -11;
erythropoietin (EPO); osteoinductive factors; interferons such as
interferon-alpha, -beta and -gamma colony stimulating factors (CSFs) such
as macrophage-CSF (M-CSF); granulocyte macrophage-CSF (GM-CSF); and
granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15,
IL-18, IL-23; a tumor necrosis factor such as TNF-alpha or TNF-beta; and
other polypeptide factors including LIF and kit ligand (KL). As used
herein, the term cytokine includes proteins from natural sources or from
recombinant cell culture and biologically active equivalents of the
native sequence cytokines.

[0042]The genetically engineered fusion molecules utilized in the current
invention are constructed using techniques well known to those of
ordinary skill in the art. The fusion molecule may have the general
designs as depicted in FIGS. 1-7. The method of preparing the fusion
molecules generally comprises the steps of: 1) preparing/obtaining a
cell/tumor targeting moiety; 2) preparing/obtaining a costimulatory
molecule and/or a chemokine and/or cytokine; 3) preparing/obtaining a
linker; 4) attaching 1) to 2) using said linker to prepare a fusion
molecule; and 5) purifying said fusion molecule. Alternatively, the
method may comprise, after step 4), step 5) preparing/obtaining a
targeting peptide; step 6) preparing/obtaining a second linker; step 7)
attaching the targeting peptide of step 5) to the fusion molecule of step
4) using said second linker to prepare a fusion molecule; and 8)
purifying said fusion molecule.

[0044]Cells suitable for replicating and for supporting recombinant
expression of protein are well known in the art. Such cells may be
transfected or transduced as appropriate with the particular expression
vector and large quantities of vector containing cells can be grown for
seeding large scale fermenters to obtain sufficient quantities of the
protein for clinical applications. Such cells may include prokaryotic
microorganisms, such as E. coli; various eukaryotic cells, such as
Chinese hamster ovary cells (CHO), NSO, 292; Yeast; insect cells; and
transgenic animals and transgenic plants, and the like. Standard
technologies are known in the art to express foreign genes in these
systems.

[0045]The pharmaceutical compositions of the present invention comprise a
genetically engineered fusion molecule of the invention and a
pharmaceutically acceptable carrier. As used herein, "pharmaceutically
acceptable carrier" means any and all solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like that are physiologically compatible. Some
examples of pharmaceutically acceptable carriers are water, saline,
phosphate buffered saline, dextrose, glycerol, ethanol and the like, as
well as combinations thereof. In many cases, it will be preferable to
include isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol, or sodium chloride in the composition. Additional
examples of pharmaceutically acceptable substances are wetting agents or
minor amounts of auxiliary substances such as wetting or emulsifying
agents, preservatives or buffers, which enhance the shelf life or
effectiveness of the antibody. Except insofar as any conventional
excipient, carrier or vehicle is incompatible with the genetically
engineered fusion molecules of the present invention, its use in the
pharmaceutical preparations of the invention is contemplated.

[0046]As used herein, the term "administration" refers to the act of
giving a drug, prodrug, or other agent, or therapeutic treatment (e.g.,
radiation therapy) to a physiological system (e.g., a subject or in vivo,
in vitro, or ex vivo cells, tissues, and organs). The compositions of
this invention may be in a variety of forms, for example, liquid,
semi-solid and solid dosage forms, such as liquid solutions (e.g.,
injectable and infusible solutions), dispersions or suspensions, tablets,
pills, powders, liposomes and suppositories. A pharmaceutical composition
of the invention is formulated to be compatible with its intended route
of administration and therapeutic application. Methods of administering
the pharmaceutical compositions of the present invention are via any
route capable of delivering the composition to a tumor cell and include,
but are not limited to, intradermal, intramuscular, intraperitoneal,
intravenous, intratumor, subcutaneous, and the like. As will be
appreciated by the skilled artisan, the route and/or mode of
administration will vary depending upon the desired results. Typical
preferred pharmaceutical compositions are in the form of injectable or
infusible solutions, such as compositions similar to those used for
passive immunization of humans. In a preferred embodiment, the
composition is administered by intravenous infusion or injection. In
another preferred embodiment, the composition is administered by
intramuscular or subcutaneous injection.

[0047]The fusion molecules of the present invention and pharmaceutical
compositions comprising them, can be administered in combination with one
or more other therapeutic, diagnostic or prophylactic agents. Additional
therapeutic agents include other anti-neoplastic, anti-tumor,
anti-angiogenic or chemotherapeutic agents. Such additional agents may be
included in the same composition or administered separately.

[0048]Therapeutic pharmaceutical compositions typically must be sterile
and stable under the conditions of manufacture and storage. Sterile
injectable solutions can be prepared by incorporating the fusion molecule
in the required amount in an appropriate solvent with one or a
combination of ingredients enumerated above, as required, followed by
filtered sterilization. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that contains a
basic dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation are
vacuum drying and freeze-drying that yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof. The proper fluidity of a solution can
be maintained, for example, by the use of a coating such as lecithin, by
the maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prolonged absorption of injectable
compositions can be brought about by including in the composition an
agent that delays absorption, for example, monostearate salts and
gelatin.

[0049]In certain embodiments, the pharmaceutical compositions active
compounds may be prepared with a carrier that will protect the
composition against rapid release, such as a controlled release
formulation, including implants, transdermal patches, and
microencapsulated delivery systems. Biodegradable, biocompatible polymers
can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic
acid, collagen, polyorthoesters, and polylactic acid. Many methods for
the preparation of such formulations are patented or generally known to
those skilled in the art. See, e.g., Sustained and Controlled Release
Drug Delivery Systems (J. R. Robinson, ed., Marcel Dekker, Inc., New
York, 1978).

[0050]In certain embodiments, the fusion molecules of the invention can be
orally administered, for example, with an inert diluent or an assimilable
edible carrier. The compound (and other ingredients, if desired) can also
be enclosed in a hard or soft shell gelatin capsule, compressed into
tablets, or incorporated directly into the subject's diet. For oral
therapeutic administration, the fusion molecules can be incorporated with
excipients and used in the form of ingestible tablets, buccal tablets,
troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To
administer a compound of the invention by other than parenteral
administration, it may be necessary to coat the compound with, or
co-administer the compound with, a material to prevent its inactivation.

[0051]Additional active compounds also can be incorporated into the
pharmaceutical compositions of the present invention. In certain
embodiments, the fusion molecule of the invention is co-formulated with
and/or co-administered with one or more additional therapeutic agents.
These agents include, without limitation, antibodies that bind other
targets, antineoplastic agents, antitumor agents, chemotherapeutic
agents, and/or other agents known in the art that can enhance an immune
response against tumor cells, e.g., IFN-β1, IL-2, IL-8, IL-12,
IL-15, IL-18, IL-23, IFN-γ, and GM-CSF. Such combination therapies
may require lower dosages of the fusion molecule as well as the
co-administered agents, thus avoiding possible toxicities or
complications associated with the various monotherapies.

[0052]The pharmaceutical compositions of the invention may include a
"therapeutically effective amount" or a "prophylactically effective
amount" of the fusion molecule of the invention. As employed herein, the
phrase "an effective amount," refers to a dose sufficient to provide
concentrations high enough to impart a beneficial effect on the recipient
thereof. The specific therapeutically effective dose level for any
particular subject will depend upon a variety of factors including the
disorder being treated, the severity of the disorder, the activity of the
specific compound, the route of administration, the rate of clearance of
the compound, the duration of treatment, the drugs used in combination or
coincident with the compound, the age, body weight, sex, diet, and
general health of the subject, and like factors well known in the medical
arts and sciences. Various general considerations taken into account in
determining the "therapeutically effective amount" are known to those of
skill in the art and are described, e.g., in Gilman et al., eds., Goodman
And Gilman's: The Pharmacological Bases of Therapeutics, 8th ed.,
Pergamon Press, 1990; and Remington's Pharmaceutical Sciences, 17th ed.,
Mack Publishing Co., Easton, Pa., 1990. A "prophylactically effective
amount" refers to an amount effective, at dosages and for periods of time
necessary, to achieve the desired prophylactic result. Typically, since a
prophylactic dose is used in subjects prior to or at an earlier stage of
disease, the prophylactically effective amount will be less than the
therapeutically effective amount.

[0053]A therapeutically effective dose can be estimated initially from
cell culture assays by determining an IC50. A dose can then be formulated
in animal models to achieve a circulating plasma concentration range that
includes the IC50 as determined in cell culture. Such information can be
used to more accurately determine useful doses in humans. Levels in
plasma may be measured, for example, by HPLC. The exact formulation,
route of administration and dosage can be chosen by the individual
physician in view of the patient's condition.

[0054]Dosage regimens can be adjusted to provide the optimum desired
response (e.g., a therapeutic or prophylactic response). For example, a
single bolus can be administered, several divided doses can be
administered over time or the dose can be proportionally reduced or
increased as indicated by the exigencies of the therapeutic situation. It
is especially advantageous to formulate parenteral compositions in dosage
unit form for ease of administration and uniformity of dosage. Dosage
unit form as used herein refers to physically discrete units suited as
unitary dosages for the mammalian subjects to be treated; each unit
containing a predetermined quantity of active compound calculated to
produce the desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms of
the present invention will be dictated primarily by the unique
characteristics of the tumor targeting moiety and the particular
therapeutic or prophylactic effect to be achieved.

[0055]An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of an antibody or antibody portion of
the invention is 0.025 to 50 mg/kg, more preferably 0.1 to 50 mg/kg, more
preferably 0.1-25, 0.1 to 10 or 0.1 to 3 mg/kg. It is to be noted that
dosage values may vary with the type and severity of the condition to be
alleviated. It is to be further understood that for any particular
subject, specific dosage regimens should be adjusted over time according
to the individual need and the professional judgment of the person
administering or supervising the administration of the compositions, and
that dosage ranges set forth herein are exemplary only and are not
intended to limit the scope or practice of the claimed composition.

[0058]This invention also relates to pharmaceutical compositions for
inhibiting abnormal cell growth in a mammal comprising an amount of a
fusion molecule of the invention in combination with an amount of a
chemotherapeutic, wherein the amounts of the fusion molecule and of the
chemotherapeutic are together effective in inhibiting abnormal cell
growth. Many chemotherapeutics are presently known in the art. In some
embodiments, the chemotherapeutic is selected from the group consisting
of mitotic inhibitors, alkylating agents, anti-metabolites, intercalating
antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes,
topoisomerase inhibitors, biological response modifiers, anti-hormones,
e.g. anti-androgens, and anti-angiogenesis agents.

[0059]The following examples are provided to describe the invention in
further detail.

Example 1

[0060]This Example describes the preparation of genetically engineered
molecules comprising a tumor targeting moiety and a chemokine. In this
example, the chemokine is fractalkine or MCP-1 and the tumor targeting
moiety is an anti-her2/neu antibody. The molecule will be constructed as
depicted in FIG. 1, with the fractalkine molecule (full length or
truncated) or MCP-1 molecule attached via a linker to the heavy chain
(HC)(e.g., IGN02/03/04/06 below) or light chain (LC)(e.g., IGN01 below)
of the antibody. The molecules in this example were prepared using
methods and techniques well known and understood by one of ordinary skill
in the art.

[0061]The preparation of the engineered molecules can be generally
described as follows: 1) the full gene sequence of interest is
synthesized by the most appropriate method, e.g., direct gene synthesis,
overlap PCR methodologies, and/or restriction-ligation techniques, for
such gene; 2) the synthesized gene sequence is incorporated into an
appropriate expression vector; 3) the expression vector is sequence
verified; 4) an expression vector is constructed to be produced and
purified in sufficient quantities from E. coli fermentation; 5) DNA
plasmids are constructed and used to perform pilot transfections of
HEK-293 Freestyle cells and the expression of the protein of interest is
monitored using reagents and/or protocols capable of detecting the
protein of interest; 6) using the information from the pilot
transfections, production scale up is performed by transfection of
HEK-293 Freestyle cells with DNA plasmids so as to produce sufficient
conditioned medium from the transfected HEK-293 Freestyle cells to
deliver the target amount of purified protein; 7) conditioned medium is
collected, concentrated, and the protein of interest purified using a
single Protein A affinity chromatography step or appropriate alternative
chromatography methods; 8) the final product is formulated in a desired
buffer and at a desired concentration (the protein concentration is
confirmed by UV absorption); and 9) the purity of the final product is
determined by SDS-PAGE.

[0062]The actual construction of six engineered molecules is depicted as
follows:

In the sequences above, the Light chain sequence would be representative
of the C sequence (amino acids 1-76), the linker sequence (amino acids
77-91) and the VL and CL sequences (amino acids 92-305) in FIG. 1. In
this example, the C sequence was the chemokine, fractalkine. The Heavy
chain sequence would be representative of the VH, CH1, CH2, and CH3
sequences in FIG. 1.

In the sequences above, the Light chain sequence would be representative
of the VL and CL sequences in FIG. 1. The Heavy chain sequence would be
representative of the C sequence (amino acids 1-76), the linker sequence
(amino acids 77-91) and the VH, CH1, CH2, and CH3 sequences (amino acids
92-542) in FIG. 1. In this example, the C sequence was the chemokine,
fractalkine.

In the sequences above, the Light chain sequence would be representative
of the VL and CL sequences in FIG. 1. The Heavy chain sequence would be
representative of the VH, CH1, CH2, and CH3 sequences (amino acids 1450),
the linker sequence (amino acids 451-465) and the C sequence (amino acids
466-541) in FIG. 1. In this example, the C sequence was the chemokine,
fractalkine.

In the sequences above, the Light chain sequence would be representative
of the VL and CL sequences in FIG. 1. The Heavy chain sequence would be
representative of the C sequence (amino acids 1-76), the linker sequence
(amino acids 77-101) and the VH, CH1, CH2, and CH3 sequences (amino acids
102-552) in FIG. 1. In this example, the C sequence was the chemokine,
fractalkine.

IGN05

[0063]The molecule tested as IGN05 was a synthetically prepared protein
having a sequence similar to that of Herceptin® (Genentech USA).
IGN05 contained no linker sequence or C sequence.

In the sequences above, the Light chain sequence would be representative
of the VL and CL sequences in FIG. 1. The Heavy chain sequence would be
representative of the C sequence (amino acids 1-76), the linker sequence
(amino acids 77-101) and the VH, CH1, CH2, and CH3 sequences (amino acids
102-552) in FIG. 1. In this example, the C sequence was the chemokine,
MCP-1.

Example 2

[0064]In this Example, the molecules of Example 1 were tested and
evaluated head-to-head with commercially available Herceptin® in a
SKBR-3 breast cancer cell binding assay and in an ADCC assay.

Cell Binding

[0065]Binding of the IGN01-06 molecules to SKBR-3 breast cancer cells was
measured by two methods. In the first method, a labeled antibody to
CX3CL1(fractalkine)(R&D Systems Cat. No. IC365P) was used. By binding to
the fractalkine moiety of the fusion proteins, this antibody acted as a
secondary antibody for the detection of the cell-bound antibody of
interest. In the second method, a labeled anti-human IgG (Jackson
ImmunResearch Cat. No. 109-096-088) was used to bind to the IgG moiety of
the fusion proteins. Both of these antibodies were used at a fixed
concentration of 5 ug/ml.

[0066]The results of these studies are shown below. Binding curves for
anti-IgG and CX3CL1 are shown in FIGS. 8 and 9, respectively. The
relative binding is shown in Table 1. The values in FIGS. 8 and 9 are
given as mean fluorescence intensity (MFI), which represents the average
fluorescence of the cells measured by FACS as a function of antibody
concentration. In Table 1, the values for maximal binding have been
normalized to the control (cells stained with secondary antibody alone)
to obtain the fold-increase in fluorescence relative to the control.

[0067]All antibody variants bound to SKBR3 cells, as indicated by the fact
that the anti-IgG secondary antibody labeled the cells at all of the
concentrations evaluated (FIG. 8). Compared to IGN05 binding to SKBR3
cells, IGN02, 03, 04, and 06 was 75-80% (FIG. 8, Table 1). The binding of
IGN01 was 29% of IGN05. With respect to the binding of anti-CX3CL1
antibody, IGN05 and 06 failed to bind (shown in FIG. 9). Because neither
IGN05 nor IGN06 have a CX3CL1 domain, they served as negative control in
this assay. The antibody bound to IGN03 to a moderate degree (50-fold
above background) while the remaining variants bound 10- to 20-fold above
background.

[0068]The IGN01-06 were also test in an ADCC assay. PBMC-mediated ADCC
against SKBR3 cells was measured using FACS. Briefly, target cells were
labeled with carboxyfluorescein, treated with IGN01-06 variant
constructs, mixed with PBMCs at an E:T of 50:1 and incubated for 4 hours
at 37° C. At that time, propidium iodide was added to label dead
cells and the samples were analyzed by FACS. Using such a methodology,
live cells fluoresce green while dead target cells fluoresce both green
and red. Importantly, each of the IGN01-06 molecules demonstrated ADCC
activity with the EC50 ranging 0.039-0.1552 μg/ml, indicating
that the addition of chemokine ligand did not cause steric hinderance to
effect ADCC.

[0069]The engineered IGN01-04 and IGN06 molecules have two sites to
interact with effector cells: 1) Fc interaction with Fc receptors; and 2)
chemokine interaction with chemokine receptor. As relates to the
fractalkine-containing molecules, the fractalkine receptor, CX3CR1, has
been shown to express in most NK and effector cytotoxic T cells.
Moreover, fractalkine will help in the migration of the cells to closer
proximity of the tumor thus providing efficient recruitment of NK and
effector cytotoxic T cells. It is possible that fractalkine-containing
molecules may overcome killer-inhibitory receptor mediated protection in
the growth of some tumors. As such, these chemokine-containing molecules
will demonstrate superior tumor killing activity of tumor cells as
compared to Herceptin® alone, including efficacy in patients
refractory to previous therapies due to Fc receptor polymorphisms in
cancer patients, thereby improving on existing tumor antigen-specific,
depleting antibody therapies.

Example 3

[0070]This Example describes the preparation of a genetically engineered
molecule comprising a tumor targeting moiety and a costimulatory
molecule. In this example, the costimulatory molecules is OX40L, 41BBL,
or CD86 and the tumor targeting moiety is an anti-her2/neu antibody. The
molecule will be constructed as depicted in FIG. 1, with the
costimulatory molecule (full length or truncated) attached via a linker
to the heavy chain (VH) of the antibody. The molecule will be prepared
using the methods described herein. The molecule will be tested and
evaluated in a cell based assay involving tumor cells and T cells and
wherein said tumor cell can not normally costimulate or activate T cells.
Importantly, because the tumor targeting moiety of the engineered
molecule will bind the tumor and display the costimulatory molecule on
the surface of the tumor, the molecule will effectively activate T cells
and help in killing the tumor, thereby improving on current
antigen-specific cytotoxic T cells mediated therapies.

[0071]All of the articles and methods disclosed and claimed herein can be
made and executed without undue experimentation in light of the present
disclosure. While the articles and methods of this invention have been
described in terms of preferred embodiments, it will be apparent to those
of skill in the art that variations may be applied to the articles and
methods without departing from the spirit and scope of the invention. All
such variations and equivalents apparent to those skilled in the art,
whether now existing or later developed, are deemed to be within the
spirit and scope of the invention as defined by the appended claims. All
patents, patent applications, and publications mentioned in the
specification are indicative of the levels of those of ordinary skill in
the art to which the invention pertains. All patents, patent
applications, and publications are herein incorporated by reference in
their entirety for all purposes and to the same extent as if each
individual publication was specifically and individually indicated to be
incorporated by reference in its entirety for any and all purposes. The
invention illustratively described herein suitably may be practiced in
the absence of any element(s) not specifically disclosed herein. Thus,
for example, in each instance herein any of the terms "comprising",
"consisting essentially of", and "consisting of" may be replaced with
either of the other two terms. The terms and expressions which have been
employed are used as terms of description and not of limitation, and
there is no intention that in the use of such terms and expressions of
excluding any equivalents of the features shown and described or portions
thereof, but it is recognized that various modifications are possible
within the scope of the invention claimed. Thus, it should be understood
that although the present invention has been specifically disclosed by
preferred embodiments and optional features, modification and variation
of the concepts herein disclosed may be resorted to by those skilled in
the art, and that such modifications and variations are considered to be
within the scope of this invention as defined by the appended claims.